Methods for determining binding affinities

ABSTRACT

The present invention relates generally to methods for screening a plurality of ligands using a biosensor device. More particularly, the present invention relates to methods for screening a plurality of antibodies from complex solutions using a surface plasmon resonance device. The methods of this invention provide kinetic and equilibrium information for such screening assays. The present invention also relates to systems for determining kinetic rate constants for such screening assays.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to methods for screening aplurality of ligands using a biosensor device. More particularly, thepresent invention relates to methods for screening a plurality ofantibodies from complex solutions using a surface plasmon resonancedevice. The methods of this invention provide kinetic and equilibriuminformation for such screening assays. The present invention alsorelates to systems for determining kinetic rate constants for suchscreening assays.

BACKGROUND OF THE INVENTION

With the advent of combinatorial libraries, there is an increasing needfor developing a method for screening a plurality of ligands thatenables the rapid and efficient determination of binding affinities.

One such need is for screening methods for determining accurate kineticand binding information for ligands in a complex solution, i.e., asolution containing an unpurified ligand. In complex solutions, theligand concentration is unknown. Screening methods in complex solutionstherefore cannot determine the kinetic association rate constant, k_(a),without a known ligand concentration. In the absence of k_(a), thebinding affinity also cannot be determined. Consequently, currentscreening methods in complex solutions are limited to providing onlyqualitative information on the presence of, or relative binding affinityof, a specific ligand in the complex solution. If accurate kinetic andbinding information are to be determined, the screening method requirespurified ligand. The requirement for using purified ligands for kineticcharacterization makes current methods time consuming and expensive.

Another need is for screening methods for determining accurate kineticand binding information for polyvalent ligands, such as antibodies. Inthe case of antibodies, a generally accepted paradigm is that antibodybinding affinity is determined by the dissociation rate constant, k_(d),and that k_(a) does not vary from one antibody to another. According tothis paradigm, antibody binding kinetics typically rely only on k_(d).Such misleading binding information makes the identification of usefulantibodies more difficult.

The present invention meets the needs referred to above by providing ascreening method for determining kinetic and binding information forinteractions between a ligand and its binding partner using a biosensordevice. The present invention also provides a method for determiningsuch information for ligands in a complex solution. The presentinvention further provides a method for screening polyvalent ligands.

SUMMARY OF THE INVENTION

The invention relates to a method for screening a plurality of ligandsusing a biosensor device. The invention also relates to methods fordetermining kinetic and equilibrium information for a plurality ofligand-binding partner interactions. The invention further relates tosystems for determining kinetic rate constants for a plurality ofligand-binding partner interactions.

In some embodiments, the invention provides a method for screening aplurality of ligands using a biosensor device, comprising the steps of(a) contacting a biorecognition surface comprising a ligand of interestwith a solution containing a binding partner; (b) collecting data forbinding of the binding partner to the ligand; (c) globally fitting thedata to a maximum response determined for a plurality of ligands bindingto the binding partner and locally fitting the data to determine kineticrate constants; and (d) calculating a binding affinity from the kineticrate constants. In some embodiments, the biorecognition surface isprepared by ligand capture from the screening solution. In someembodiments, the ligand of interest is selected from the groupconsisting of proteins, including, but not limited to, antibodies,receptors and enzymes; nucleic acids; carbohydrates; lipids; and smallmolecules. In some embodiments, the binding partner is selected from thegroup consisting of proteins, including, but not limited to, antigens,receptors and enzymes; nucleic acids; carbohydrates; lipids; and smallmolecules. In some embodiments, the biosensor device is selected fromthe group consisting of an evanescent wave, total internal reflectionfluorescence and surface plasmon resonance devices.

In some embodiments, the invention provides a method for screening aplurality of ligands from a complex solution using a biosensor device,comprising the steps of (a) contacting a biorecognition surfacecomprising a ligand of interest with solution containing a bindingpartner, wherein the biorecognition surface is prepared by ligandcapture from the complex solution; (b) collecting data for binding ofthe binding partner to the ligand; (c) globally fitting the data to amaximum response determined for a plurality of ligands binding to thebinding partner and locally fitting the data to determine kinetic rateconstants; and (d) calculating a binding affinity from the kinetic rateconstants. In some embodiments, the ligand of interest is selected fromthe group consisting of proteins, including, but not limited to,antibodies, receptors and enzymes; nucleic acids; carbohydrates; lipids;and small molecules. In some embodiments, the binding partner isselected from the group consisting of proteins, including, but notlimited to, antigens, receptors and enzymes; nucleic acids;carbohydrates; lipids; and small molecules. In some embodiments, thebiosensor device is selected from the group consisting of an evanescentwave, total internal reflection fluorescence and surface plasmonresonance devices.

In some embodiments, the invention provides a method for screening aplurality of antibodies from complex solutions using a surface plasmonresonance device, comprising the steps of (a) contacting abiorecognition surface comprising an antibody with solution containingan antigen, wherein the biorecognition surface is prepared by antibodycapture from the complex solution; (b) collecting data for binding ofthe antigen to the antibody; (c) globally fitting the data to a maximumresponse determined for a plurality of antibodies binding to the antigenand locally fitting the data to determine kinetic rate constants; and(d) calculating a binding affinity from the kinetic rate constants.

In some embodiments, the invention provides a method for determiningkinetic rate constants for a plurality of ligand-binding partnerinteractions using a biosensor device, comprising the steps of (a)contacting a biorecognition surface comprising the ligand with asolution containing the binding partner; (b) collecting data for bindingof the binding partner to the ligand; and (c) globally fitting the datato a maximum response determined for a plurality of ligands binding tothe binding partner and locally fitting the data to determine kineticrate constants. In some embodiments, the ligand is selected from thegroup consisting of proteins, including, but not limited to, antibodies,receptors and enzymes; nucleic acids; carbohydrates; lipids; and smallmolecules. In some embodiments, the binding partner is selected from thegroup consisting of proteins, including, but not limited to, antigens,receptors and enzymes; nucleic acids; carbohydrates; lipids; and smallmolecules. In some embodiments, the biosensor device is selected fromthe group consisting of an evanescent wave, total internal reflectionfluorescence and surface plasmon resonance devices.

In some embodiments, the invention provides a method for determiningkinetic rate constants for a plurality of antibody-antigen interactionsusing a biosensor device, comprising the steps of (a) contacting abiorecognition surface comprising an antibody with a solution containingthe antigen; (b) collecting data for binding of the antigen to theantibody; and (c) globally fitting the data to a maximum responsedetermined for a plurality of antibodies binding to the antigen andlocally fitting the data to determine kinetic rate constants. In someembodiments, the biosensor device is selected from the group consistingof an evanescent wave, total internal reflection fluorescence andsurface plasmon resonance devices. In some embodiments, the antibodycapture is from a complex solution. In some embodiments, the antibodycapture is from a pure solution.

In some embodiments, the invention provides a system for determiningkinetic rate constants for a plurality of ligand-binding partnerinteractions using a biosensor device, comprising (a) a biorecognitionsurface comprising a ligand; (b) a means for processing data for bindinginteractions between the ligand and the binding partner; and (c) a meansfor globally fitting the data to a maximum response determined for aplurality of ligands binding to the binding partner and locally fittingthe data to determine the rate constants. In some embodiments, thebiorecognition surface is prepared by ligand capture. In someembodiments, the biorecognition system is prepared by ligand capturefrom a complex solution. In some embodiments, the biorecognition systemis prepared by ligand capture from a pure solution. In some embodiments,the ligand is selected from the group consisting of proteins, including,but not limited to, antibodies, receptors and enzymes; nucleic acids;carbohydrates; lipids; and small molecules. In some embodiments, thebinding partner is selected from the group consisting of antigens,proteins, including, but not limited to, antigens, receptors andenzymes; nucleic acids; carbohydrates; lipids; and small molecules. Insome embodiments, the biosensor device is selected from the groupconsisting of an evanescent wave, total internal reflection fluorescenceand surface plasmon resonance devices.

In some embodiments, the invention provides a system for determiningkinetic rate constants for a plurality of antibody-antigen interactionsusing a biosensor device, comprising (a) a biorecognition surfacecomprising an antibody; (b) a means for processing data for bindinginteractions between an antigen and the antibody; and (c) a means forglobally fitting the data to a maximum response determined for aplurality of antibodies binding to the antigen and locally fitting thedata to determine the rate constants. In some embodiments, thebiorecognition system is prepared by antibody capture from a complexsolution. In some embodiments, the biorecognition system is prepared byantibody capture from a pure solution. In some embodiments, thebiosensor device is selected from the group consisting of an evanescentwave, total internal reflection fluorescence and surface plasmonresonance devices.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 shows a typical set of sensorgrams for capturing antibody to aprotein A immobilized surface and for binding of antigen to theantibody-captured protein A surface. Antibody was captured by animmobilized protein A surface (A). The antibody-captured protein Asurface was washed for 10 minutes to stabilize the baseline signal (B).A buffer injection was collected to gather information about thebackground surface decay (C). Finally, a single concentration of antigenwas injected over the surface (D). One protein A surface served as thecontrol (data shown were reference subtracted). The box indicates theantibody capture level at the time of antigen injection.

FIG. 2 shows a typical set of sensorgrams for normalizing the backgrounddecay of an antibody-captured protein A surface.

FIG. 3 shows typical raw and normalized sensorgrams of antigen bindingto antibody-captured protein A surface.

FIG. 4 shows global analysis of normalized sensorgrams.

FIG. 5 shows a typical set of sensorgrams of antigen binding toantibody-captured protein A surfaces in a high throughput screen.

FIG. 6 shows a plot of antibody capture level versus observed antigenbinding response.

FIG. 7 shows a typical set of sensorgrams of antigen binding toantibody-captured protein A surfaces using a range of antigenconcentrations.

FIG. 8 shows a plot of antibody affinities determined from single ormultiple concentrations of antigen.

FIG. 9 shows the capture-coupling method for immobilizing antibody to aprotein A surface.

FIG. 10 shows normalized data for antigen binding to antibodyimmobilized using the capture-coupling method.

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application, including the definitions, will control. Also,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Allpublications, patents and other references mentioned herein areincorporated by reference.

Although methods and materials similar or equivalent to those describedherein can be used in practice or testing of the present invention,exemplary suitable methods and materials are described below. Thematerials, methods and examples are illustrative only, and are notintended to be limiting. Other features and advantages of the inventionwill be apparent from the detailed description and from the claims.

Throughout the specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

In order to further define this invention, the following terms anddefinitions are herein provided.

As used herein, the term “biosensor device” means an analytical devicecomprising a biorecognition surface. Such a device typically produces asignal in response to a binding interaction at the biorecognitionsurface. The term includes, but is not limited to, evanescent wave,total internal reflection fluorescence (“TIRF”) and surface plasmonresonance (“SPR”) devices.

As used herein, the term “biorecognition surface” means a solid supportcomprising a ligand of interest.

As used herein, the term “solid support” means a material in thesolid-phase that interacts with reagents in the liquid phase byheterogeneous reactions. Solid-supports can be derivatized with ligandsby covalent or non-covalent bonding through one or more attachmentsites, thereby “immobilizing” the ligand to the solid-support. The termincludes, but is not limited to, glass surfaces, metal-coated glasssurfaces, such as gold-coated, and modifications thereof. Suitablemodifications include, but are not limited to, interactive surfacelayers. Examples of interactive surface layers include, but are notlimited to, carboxymethyl-dextran hydrogel, alkoxy silanes (e.g.,BIO-CONEXT™ from United Chemical Technologies, Inc.) and self-assembledmonolayers (“SAMs”).

As used herein, the term “complex solution” means a solution comprisingan unpurified ligand of interest. The term includes, but is not limitedto, cell culture media, hybridoma supernatants, ascites fluid, serum,cell lysates or fractions thereof, column effluents, mixtures of ligandwith other substances and the like.

As used herein, the term “unpurified ligand” means a ligand with lessthan about 90% purity.

As used herein, the term a “pure solution” means a solution with greaterthan about 90% purity.

As used herein, the term “ligand capture” means the process by which anagent immobilized on a solid support (“a capture agent”) captures anyligand present in a solution. The term includes, but is not limited to,antibody capture. Capture agents include, but are not limited to,protein A and antibodies, such as anti-isotype antibodies.

As used herein, the term “sensorgram” means a plot of response (measuredin “resonance units” or “RU”) as a function of time. The responsecorresponds to the amount of material that binds to a sensor surface. Anincrease of 1000 RU corresponds to an increase of mass on the sensorsurface of approximately 1 ng/mm². “R_(max)” means the responsecorresponding to the maximum binding capacity of the sensor surface.

As used herein, the term “association” means the step where ligand boundto a sensor surface interacts with a binding partner in solution. Thisstep is indicated on the sensorgram by an increase in RU as the bindingpartner binds to the surface-bound ligand.

As used herein, the term “dissociation” means the step where the flow ofbinding partner is replaced by, for example, a flow of buffer. This stepis indicted on the sensorgram by a decrease in RU over time as bindingpartner dissociates from the surface-bound ligand.

As used herein, the terms “ligand of interest” and “binding partner”mean members of a specific binding pair. Examples of ligands include,but are not limited to, proteins, including, but not limited to,antibodies, receptors and enzymes; nucleic acids; carbohydrates; lipids;and small molecules. Examples of binding partners include, but are notlimited to, proteins, including, but not limited to, antigens, receptorsand enzymes; nucleic acids; carbohydrates; lipids; and small molecules.

As used herein, the term “antibody” means an intact immunoglobulin or afunctional binding fragment thereof. Antibodies of this invention can beof any isotype or class (e.g., M, D, G, E and A) or any subclass (e.g.,G1-4, A1-2) and can have either a kappa (κ) or lambda (λ) light chain.

As used herein, the term “F_(c)” means a portion of the heavy chainconstant region of an antibody that is produced by papain digestion.

As used herein, the term “antigen” means a molecule containing one ormore epitopes that will stimulate a host's immune system to make ahumoral and/or cellular antigen-specific response.

As used herein, the term “epitope” means the site on an antigen to whicha specific antibody molecule binds.

The following abbreviations are also used herein SPR, surface plasmonresonance; TIRF, total internal reflection fluorescence; CM-dextran,carboxymethyl-dextran; k_(a), association rate constant; k_(d),dissociation rate constant; RU, response units; and SAMs, self-assembledmonolayers.

Methods for Screening Solutions

In one aspect, the present invention provides methods for the rapid andefficient screening of a plurality of ligand samples using a biosensordevice to determine intrinsic kinetic and binding information forligand-binding partner interactions. The number of samples can be anynumber from 1 up to the limits of the biosensor devise, i.e., at least10, at least 30, at least 50, at least 75, at least 100, at least 125,at least 150, at least 175, etc.

The methods of the invention can be utilized with any ligand. The ligandmay be monovalent, divalent or polyvalent. Exemplary ligands that can beused in the methods of the invention include, but are not limited to,proteins, including, but not limited to, antibodies, receptors andenzymes; nucleic acids; carbohydrates; lipids; and small molecules.Similarly, the methods of the invention can be used with any bindingpartner. The binding partner can be monovalent, bivalent or polyvalent.Exemplary binding partners that can be used in the methods of theinvention include, but are not limited to, proteins, including, but notlimited to, antigens, receptors and enzymes; nucleic acids;carbohydrates; lipids; and small molecules.

In some embodiments, the ligand is purified. In other embodiments theligand is unpurified. In some embodiments, the unpurified ligand is in acomplex solution. Exemplary complex solutions are cell culture media,hybridoma supernatants, ascites fluid, serum, cell lysates or fractionsthereof, column effluents, mixtures of ligand with other substances andthe like.

In a preferred embodiment, the ligand is an antibody and the bindingpartner is an antigen. In some embodiments, the antibody is purified. Inother embodiments, the antibody is unpurified. In some embodiments, theunpurified antibody is in a complex solution, such as (but not limitedto), cell culture media, hybridoma supernatants, ascites fluid, serum,cell lysates or fractions thereof, column effluents, mixtures of ligandwith other substances and the like.

Any suitable solid support can be used to generate a biorecognitionsurface for use in a biosensor device. In a preferred embodiment, thesolid support is glass. In some embodiments, the solid support isgold-coated glass. Preferably the solid support is coated with aninteractive surface layer. Exemplary interactive surface layers arecarboxymethyl-dextran hydrogel, alkoxy silanes and self-assembledmonolayers (“SAMs”).

In a preferred embodiment, the biosensor device is an SPR device, suchas a BIACORE device.

In some embodiments, the ligand is immobilized directly to theinteractive layer, such as by amine coupling to carboxymethyl-dextran.However, where a large number of samples are to be analyzed,immobilization of the ligand to a composition that provides an easilyregeneratable surface is preferred. In this way, the biorecognitionsurface can be quickly and easily regenerated for repeated use withnumerous samples.

In embodiments using unpurified ligand samples, particularly where theligand is in a complex solution, a capturing agent is used to immobilizethe ligand onto the surface of a solid support to generate abiorecognition surface. The choices of capture agent for a ligand ofinterest are well-known in the art. The selection of an appropriatecapture agent for use in the methods of the invention is well within theskill of the art.

In a preferred embodiment, the samples are antibodies in hybridomasupernatant. If the sample contains an IgG antibody, for example,Protein A or an anti-IgG antibody can be used as a capture agent.Capture agents for other antibody isotypes are well known in the art,e.g., anti-isotype antibodies. Immobilizing the antibody on the solidsurface using a capturing agent, in addition to permitting the screeningof unpurified antibodies, provides a number of additional advantagesincluding providing antibodies immobilized in a more homogeneousorientation and thus providing more uniform biorecognition surfaces thanis provided by direct immobilization, and permitting rapid regenerationof the solid surface between batches of samples. Further, byimmobilizing the antibody, one can utilize known concentrations ofantigen in the binding step. Because the antigen concentration is known,the association rate (k_(a)) can be determined. As a result, one candetermine a more accurate binding affinity, i.e., one based on bothassociation and dissociation rates, for each antibody.

According to the method, the biorecognition surface comprising theligand is contacted with a single concentration of binding partnersolution and the biosensor device collects data for the bindinginteraction between the ligand and the binding partner. In someembodiments, the biosensor device is an SPR device, the ligand is apurified ligand that is directly immobilized on a solid support. In someembodiments, the biosensor device is an SPR device, the ligand is apurified ligand that is captured by a capture agent immobilized on asolid support. In either of these embodiments, the ligand can be anantibody.

Contacting each sample with the same single concentration of bindingpartner instead of contacting each sample with a range of differentconcentrations allows the rapid and efficient analysis of large numbersof samples.

According to the methods of the invention, the collected binding data isthen processed to correct the binding signal for general noise,non-specific binding of the binding partner to the solid support andbaseline drift.

In the methods of the invention, kinetic analyses are performed byglobally fitting the processed binding data. In the global fitting, thebinding data from multiple samples is fit to a single binding-site modeland a single “global” Rmax is determined. According to the globalfitting analysis, k_(a) and k_(d) are permitted to be “local” parametersthat are determined using a constant Rmax, i.e., the global Rmax.Binding affinity is then determined for each sample using the kineticrate constants.

As will be readily understood, the method according to this aspect ofthe invention is well suited for screening large collections of ligands.High binding affinity ligands identified by this method may be furtherevaluated in higher resolution experiments, e.g., in experimentsutilizing multiple concentrations of binding partners.

System for Determining Kinetic Rate Constants for Ligand-Binding PartnerInteractions

In a further aspect, the present invention provides a system fordetermining kinetic rate constants for ligand-binding partnerinteractions using a biosensor device. The system comprises (a) abiorecognition surface comprising a ligand; (b) a means for processingdata for binding interactions between the ligand and a binding partner;and (c) a means for globally fitting the data to a maximum responsedetermined for a plurality of ligand binding to the binding partner andlocally fitting the data to determine the rate constants.

In some embodiments, the biosensor device is an SPR device. In someembodiments, the biosensor device is an evanescent wave device. In someembodiments, the biosensor device is a TIRF device.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLES

In these experiments, we screened a panel of monoclonal antibodysupernatants to determine the binding affinities of the antibodies usingSPR.

Example 1 Preparation of Biorecognition Surfaces

We prepared a biorecognition surface by first immobilizing Immunopure®Protein A (Pierce, Rockford, Ill.) to CM5 sensor chips (BIACORE AB,Uppsala, Sweden) in a BIACORE 2000 or 3000 instrument (FIG. 1) asfollows.

In the BIACORE instrument, the sensor chips were pre-conditioned inwater at a flow rate of 100 μL/min by applying two consecutive 20-μLpulses of 50 mM NaOH, 0.1% HCl (v/v) and 0.1% SDS. The individual flowcells were equilibrated with 10 mM HEPES buffer containing 150 mM NaCland 0.005% P-20 Surfactant (BIACORE AB), pH 7.4 (“HBSP running buffer”)at a flow rate of 20 μL/min. Next, a solution of 70 μL of 50 mMN-hydroxysuccinimide (“NHS;” BIACORE AB) and 70 μL of 200 mM1-(3-dimethylaminopropyl)-ethylcarbodiimide hydrochloride (“EDC;”BIACORE AB) was injected over the flow cells to activate the CM-dextran.Johnsson et al., Anal Biochem, 198, pp. 268-277 (1991). A solution ofprotein A (reconstituted in water to 5 mg/mL and diluted to 150 μg/mL in10 mM sodium acetate at pH 5.0) was flowed across the flow cells for 7min at a flow rate of 20 μL/min followed by a 140 μL injection of 1 Msodium ethanolamine.HCl at pH 8.5 (BIACORE AB). The immobilized proteinA surfaces were immediately conditioned by three injections of 100 mMH₃PO₄ for 6 sec. Johnsson et al., Biotechniques, 11, pp. 620-627 (1991).The typical immobilization level of protein A was 6,000 to 8,000 RU.

In other experiments, we immobilized goat anti-IgG (Fc sp.) followingthe procedure described above for immobilizing protein A. In thisprocedure, we prepared a solution of IgG in 10 mM sodium acetate at pH5.0 (100 μg/mL). The typical immobilization level of goat anti-IgG (Fcsp.) was 3,500 to 4,500 RU.

Example 2 Ligand Capture

We used the protein A or goat anti-IgG (Fc sp.) immobilized on thesensor chip surface to capture monoclonal antibodies from hybridomasupernatants (FIG. 1A) as follows. Myszka, J. Mol. Recognit., 12, pp.279-284 (1999) and Svensson et al., Eur. J. Biochem., 258, pp. 890-896(1998). Buffer (10 mM HEPES, 150 mM NaCl, 0.005% polysorbate-20, pH 7.4)containing 12 mg/mL of each BSA (Sigma, St. Louis, Mo.) and solublecarboxymethyl-dextran (“CM-dextran;” Fluka BioChemika) was flowed acrossthe individual flow cells at a flow rate of 100 μL/min. Three hybridomasupernatant solutions containing antibodies of interest were diluted1/25 in the same buffer and then separately injected over three flowcells for 5 min at a flow rate of 50 μL/min. The fourth flow cell wasnot exposed to an antibody solution and therefore, served as a control.The antibody-captured protein A surface was then washed for 10 min at aflow rate of 50 μL/min to remove any non-specific components adhering tothe surface (FIG. 1B).

Example 3 Screening of Binding Partner

We screened the antibody-captured protein A surfaces for binding toantigen (FIG. 1D) as follows. Prior to antigen injection, a solution ofbuffer was injected to determine baseline drift caused by the decay ofthe antibody-captured protein A surface (FIG. 1C). Antigen binding wasmeasured by flowing an antigen at a predetermined concentration acrossthe individual flow cells for 1 min at a flow rate of 100 μL/min andthen reintroducing the buffer for 5 min to initiate dissociation. Afterdissociation, the protein A surface was regenerated by injecting 10 μLof 100 mM H₃PO₄ for 12 sec. After regeneration, we repeated the antibodycapture procedure described in Example 2 using three supernatants fromthe panel at a time until the entire panel was screened.

To determine the concentration of antigen to be used in theantigen-binding step, we first assessed the quality of antigen bindingto the antibody-captured protein A surfaces using three differentconcentrations of antigen and from 3-6 hybridoma supernatants. Anantigen concentration was chosen that resulted in a response having aminor to significant curvature in the association phases for the testedhybridoma supernatant solutions. This variation in curvature indicateseither a range of association rates for the different antibodies or aconcentration dependence of antigen binding to the same antibody.

When the response was asymptotic to the theoretical response level(calculated by multiplying the mass ratio of antigen to antibody byRU_(captured)), we repeated the antigen binding cycle using an antigenconcentration diluted 3-fold (using further dilutions if necessary).

When the response had a change in RU less than 10 (i.e., not discernablefrom noise), we repeated the capture-binding cycle using a five-fold ormore higher concentration of hybridoma supernatant solution. Byincreasing the antibody concentration, we increased the amount ofantibody captured by the protein A surface and, thus, the amount ofantigen bound.

Example 4 Data Processing

When sensorgrams for all members of the panel were obtained, weprocessed the data from the sensorgrams to correct the binding signalfor general noise, non-specific binding of antigen to the protein Asurface and baseline drift as follows (FIG. 2 and FIG. 3).

We first corrected the binding signal for bulk refractive index changesand non-specific binding by subtracting the signal for antigen flowedacross the control flow cell which had a protein A immobilized surfacebut no antibody (“the control signal”) from the signal for antigenflowed across antibody-captured protein A surface.

Next, we aligned the signals and subtracted blank buffer injections toaccount for baseline drift (i.e., background decay of theantibody-captured protein A surface) (FIG. 2). Baseline drift wasaccounted for by subtracting the signal for the blank buffer injection,indicated by FIG. 1C, from that for the antigen injection, indicated byFIG. 1D.

Alternatively, we followed a modified protocol for subtracting baselinedrift. In this protocol, an antigen binding signal (“signal 1”) wasobtained as described above, but omitting the buffer injectionpreceeding the antigen injection. The protein A surface was thenregenerated. Next, the antibody-captured protein A surface was preparedagain following the procedure described above and a signal (“signal 2”)was obtained by flowing buffer alone across the surface for theremainder of the cycle. Baseline drift was accounted for by subtractingsignal 2 from signal 1.

Lastly, we normalized the data with respect to the amount of antibodycaptured (FIG. 3). The normalization step consists of: (1) determiningthe antibody capture signal by averaging the signal obtained during the20 seconds prior to antigen injection (indicated with a box in FIG. 1);(2) dividing the antigen binding signal by the antibody capture signal;and (3) multiplying the quotient by the mass ratio of antibody toantigen. These processing steps may be performed using BIACORE'sBiaevaluations software.

Example 5 Globally Fitting the Binding Data

We determined the binding affinities for each of the antibodies in thepanel. First, we performed kinetic analyses by globally fitting thebinding data. Using a 1:1 binding model in the software program CLAMP(www.cores.utah.edu/interaction), we simultaneously fit six normalizedsensorgrams for the binding of a single antigen to differentantibody-captured protein A surfaces. Myszka et al., Trends Biochem.Sci., 23, pp. 149-150 (1998). We determined a global R_(max) value byfirst selecting six sensorgrams representing a range of antigen bindingresponses. Second, we globally fitted those sensorgrams holding R_(max)constant and locally fitted the association and dissociation rateconstants. In subsequent fits, we applied the global R_(max) value andthe fixed antigen concentration to locally fit the association anddissociation rate constants.

Example 6 Screening Analysis

We screened 150 mAbs from hybridoma supernatants in three panels forbinding to an antigen following the protocols of Examples 1 through 5(FIG. 5). Table 1 shows the kinetic rate constants for antibody-antigeninteractions determined in the screen. We calculated the antigen bindingaffinities of the antibodies from the k_(a) and k_(d) parameters fromthe processed sensorgrams.

Example 7 Medium Resolution Analysis

We tested the reliability of the screening procedures of Examples 1-6 byre-assaying 24 mAbs in the panel using a range of antigen concentrationsfor each Ab (“medium resolution analysis”) (FIG. 7). A 100 μL solutionof supernatant containing an antibody of interest (diluted 25-fold inHBSP containing 100 mg/mL BSA) was flowed across a protein A surface ata flow rate of 20 μL/min. The antibody-captured protein A surface waswashed for 6 min at a flow rate of 50 μL/min. Next, a 50 μL solution ofantigen (0, 22.2, 66.6, 200.0 or 600.0 nM) was injected. Thedissociation phase for most of the mAbs was monitored for 120 sec andthe protein A surface was regenerated by a 10 μL injection of H₃PO₄ formost Abs. However, we found that, in the single antigenic concentrationscreen, six out of thirty mAbs from Panel 2 supernatants displayeddissociation rate constants of about 10⁻⁵ s⁻¹, giving rise to verystable Ab-Ag complexes. For these mAbs, we monitored the dissociationphase for 10 min in the medium resolution screen.

We processed the data as described in Example 4. We analyzed theprocessed data using a 1:1 interaction model in CLAMP. We normalized thedata by dividing R_(max) calculated from the antigen-binding kineticsfor each mAb. Table 1 shows the kinetic rate constants obtained in thismedium resolution analysis. TABLE 1 Kinetic parameters of Panels 1, 2,and 3 antigen- antibody interactions from the low and medium-resolutionanalysis. Association rates Dissociation rates (M⁻¹ s⁻¹) (s⁻¹) Low-Medium- Low- Medium- Ab resolution resolution resolution resolutionSupernatants Panel 1 1 1.90E+05 3.06E+05 1.00E−05 2.50E−04 2 2.50E+052.51E+05 1.00E−03 6.46E−04 3 2.03E+05 2.01E+05 8.05E−04 7.12E−04 41.81E+05 2.48E+05 8.41E−04 9.20E−04 5 1.83E+05 2.26E+05 1.14E−039.79E−04 6 1.88E+05 1.96E+05 8.80E−04 9.63E−04 7 1.69E+05 1.33E+059.86E−04 6.62E−04 8 1.60E+05 1.63E+05 9.90E−04 8.49E−04 9 1.59E+051.52E+05 8.97E−04 8.41E−04 10 1.59E+05 1.64E+05 9.56E−04 9.67E−04 111.38E+05 1.50E+05 1.30E−03 8.94E−04 12 1.67E+05 1.67E+05 1.12E−031.04E−03 13 1.67E+05 1.62E+05 1.13E−03 1.02E−03 14 1.81E+05 1.68E+051.30E−03 1.07E−03 15 1.41E+05 1.66E+05 1.21E−03 1.06E−03 16 1.91E+051.51E+05 1.17E−03 9.85E−04 17 1.86E+05 1.53E+05 1.23E−03 1.07E−03 182.02E+05 1.35E+05 1.10E−03 9.76E−04 19 2.10E+05 1.46E+05 8.99E−041.06E−03 20 1.72E+05 1.19E+05 1.16E−03 9.58E−04 21 1.28E+05 9.46E+041.07E−03 8.56E−04 22 2.56E+05 2.19E+05 1.46E−03 2.14E−03 23 1.93E+051.56E+05 1.12E−03 1.68E−03 24 2.37e+05  6.86E+04 6.00E−02 2.30E−02Supernatants Panel 2 1 5.0 × 10⁵ 8.6 × 10⁵ 5.2 × 10⁻⁴ 1.1 × 10⁻⁵ 2 5.0 ×10⁵ 5.0 × 10⁵ 2.9 × 10⁻⁴ 2.9 × 10⁻⁵ 3 8.6 × 10⁵ 9.5 × 10⁵ 9.3 × 10⁻⁴ 1.7× 10⁻⁵ 4 2.1 × 10⁵ 2.1 × 10⁵ 3.6 × 10⁻⁵ 1.6 × 10⁻⁵ 5 2.1 × 10⁵ 1.9 × 10⁵2.7 × 10⁻⁴ 3.4 × 10⁻⁵ 6 8.7 × 10⁵ 5.0 × 10⁵ 3.6 × 10⁻⁴ 3.4 × 10⁻⁵Supernatants Panel 3 1  1.0E+07  7.3E+08  4.3E−04  5.9E−04 2  5.4E+06 1.5E+08  3.4E−04  3.3E−04 3  9.2E+06  4.1E+08  4.1E−04  1.4E−03 4 1.4E+07  3.7E+08  9.8E−04  1.3E−03 5  1.1E+07  4.2E+08  5.6E−04 1.8E−03 6  7.5E+07  1.1E+08  3.9E−03  5.8E−04 7  1.1E+07  3.7E+08 7.4E−04  2.2E−03 8  1.3E+07  1.5E+08  4.5E−04  1.1E−03 9  1.3E+07 2.6E+07  6.6E−04  3.7E−04

Example 8 Capture-Coupling

We also evaluated select, highly stable antigen-antibody interactionsusing antibody-coupled protein A surfaces (FIG. 9). A solution of NHSand EDC was injected over a protein A-CM-dextran surface for 7 min at aflow rate of 20 μL/min. A solution of antibody was flowed across theflow cells at a flow rate of 5-10 μL/min followed by an injection of 1 Msodium ethanolamine.HCl at pH 8.5. Antigen binding was measured byflowing a solution of antigen (0, 2.4, 7.4, 22.2, 66.7 or 200 nM) inHBSP containing 200 μg/mL BSA across the antibody-coupled protein Asurface. The antibody-coupled protein A surfaces were regenerated using10 mM H₃PO₄. We processed the data as described above and then fit theprocessed data to a 1:1 interaction model using CLAMP (FIG. 10). Wedetermined kinetic rate constants and binding affinities.

1. A method for screening a plurality of ligands using a biosensor device, comprising the steps of: a. contacting a biorecognition surface comprising a ligand of interest with a solution containing a binding partner; b. collecting data for binding of the binding partner to the ligand; c. globally fitting the data to a maximum response determined for a plurality of ligands binding to the binding partner and locally fitting the data to determine kinetic rate constants; and d. calculating a binding affinity from the kinetic rate constants.
 2. The method according to claim 1, wherein the biorecognition surface is prepared by ligand capture from the screening solution.
 3. The method according to claim 1 or 2, wherein the ligand of interest is selected from the group consisting of proteins, antibodies, receptors, enzymes, nucleic acids, carbohydrates, lipids and small molecules.
 4. The method according to claim 1 or 2, wherein the binding partner is selected from the group consisting of proteins, antigens, receptors, enzymes, nucleic acids, carbohydrates, lipids and small molecules.
 5. The method according to claim 1 or 2, wherein the biosensor device is selected from the group consisting of an evanescent wave, total internal reflection fluorescence and surface plasmon resonance devices.
 6. A method for screening a plurality of ligands from a complex solution using a biosensor device, comprising the steps of: a. contacting a biorecognition surface comprising a ligand of interest with a solution containing a binding partner, wherein the biorecognition surface is prepared by ligand capture from the complex solution; b. collecting data for binding of the binding partner to the ligand; c. globally fitting the data to a maximum response determined for a plurality of ligands binding to the binding partner and locally fitting the data to determine kinetic rate constants; and d. calculating a binding affinity from the kinetic rate constants.
 7. The method according to claim 6, wherein the ligand is selected from the group consisting of proteins, antibodies, receptors, enzymes, nucleic acids, carbohydrates, lipids and small molecules.
 8. The method according to claim 6, wherein the binding partner is selected from the group consisting of proteins, antigens, receptors, enzymes, nucleic acids, carbohydrates, lipids and small molecules.
 9. The method according to claim 6, wherein the biosensor device is selected from the group consisting of an evanescent wave, total internal reflection fluorescence and surface plasmon resonance devices.
 10. A method for screening a plurality of antibodies from complex solutions using a surface plasmon resonance device, comprising the steps of: a. contacting a biorecognition surface comprising antibody with a solution containing an antigen, wherein the biorecognition surface is prepared by antibody capture from the complex solution; b. collecting data for binding of the antigen to the antibody; c. globally fitting the data to a maximum response determined for a plurality of antibodies binding to the antigen and locally fitting the data to determine kinetic rate constants; and d. calculating a binding affinity from the kinetic rate constants.
 11. A method for determining kinetic rate constants for a plurality ligand-binding partner interactions using a biosensor device, comprising the steps of: a. contacting a biorecognition surface comprising the ligand with a solution containing the binding partner; b. collecting data for binding of the binding partner to the ligand; and c. globally fitting the data to a maximum response determined for a plurality of ligands binding to the binding partner and locally fitting the data to determine kinetic rate constants.
 12. The method according to claim 11, wherein the ligand is selected from the group consisting of proteins, antibodies, receptors, enzymes, nucleic acids, carbohydrates, lipids and small molecules.
 13. The method according to claim 11, wherein the binding partner is selected from the group consisting of proteins, antigens, receptors, enzymes, nucleic acids, carbohydrates, lipids and small molecules.
 14. The method according to claim 11, wherein the biosensor device is selected from the group consisting of an evanescent wave, total internal reflection fluorescence and surface plasmon resonance devices.
 15. A method for determining kinetic rate constants for a plurality of antibody-antigen interactions using a biosensor device, comprising the steps of: a. contacting a biorecognition surface comprising an antibody with a solution containing the antigen; b. collecting data for binding of the antigen to the antibody; and c. globally fitting the data to a maximum response determined for a plurality of antibodies binding to the antigen and locally fitting the data to determine kinetic rate constants.
 16. The method according to claim 15, wherein the biosensor device is selected from the group consisting of an evanescent wave, total internal reflection fluorescence and surface plasmon resonance devices.
 17. The method according to claim 15, wherein the antibody capture is from a complex solution.
 18. The method according to claim 15, wherein the antibody capture is from a pure solution.
 19. A system for determining kinetic rate constants for a plurality of ligand-binding partner interactions using a biosensor device, comprising: a. a biorecognition surface comprising a ligand; b. a means for processing data for binding interactions between the ligand and the binding partner; and c. a means for globally fitting the data to a maximum response determined for a plurality of ligands binding to the binding partner and locally fitting the data to determine the rate constants.
 20. The system according to claim 19, wherein the biorecognition surface is prepared by ligand capture.
 21. The system according to claim 20, wherein the ligand capture is from a complex solution.
 22. The system according to claim 20, wherein the ligand capture is from a pure solution.
 23. The system according to claim 19, wherein the biosensor device is selected from the group consisting of an evanescent wave, total internal reflection fluorescence and surface plasmon resonance devices.
 24. A system for determining kinetic rate constants for a plurality of antibody-antigen interactions using a biosensor device, comprising: a. a biorecognition surface comprising an antibody; b. a means for processing data for binding interactions between an antigen and the antibody; and c. a means for globally fitting the data to a maximum response determined for a plurality of antibodies binding to the antigen and locally fitting the data to determine the rate constants.
 25. The system according to claim 24, wherein the biorecognition surface is prepared by antibody capture.
 26. The system according to claim 25, wherein the antibody capture is from a complex solution.
 27. The system according to claim 25, wherein the antibody capture is from a pure solution.
 28. The system according to claim 24 or 25, wherein the biosensor device is selected from the group consisting of an evanescent wave, total internal reflection fluorescence and surface plasmon resonance devices. 